Siponimod treatment response shows partial BDNF dependency in multiple sclerosis models

So far, only a small number of medications are effective in progressive multiple sclerosis (MS). The sphingosine-1-phosphate-receptor (S1PR)-1,5 modulator siponimod, licensed for progressive MS, is acting both on peripheral immune cells and in the central nervous system (CNS). So far it remains elusive, whether those effects are related to the neurotrophin brain derived neurotrophic factor (BDNF). We hypothesized that BDNF in immune cells might be a prerequisite to reduce disease activity in experimental autoimmune encephalomyelitis (EAE) and prevent neurotoxicity. MOG35–55 immunized wild type (WT) and BDNF knock-out (BDNFko) mice were treated with siponimod or vehicle and scored daily in a blinded manner. Immune cell phenotyping was performed via flow cytometry. Immune cell infiltration and demyelination of spinal cord were assessed using immunohistochemistry. In vitro, effects on neurotoxicity and mRNA regulation were investigated using dorsal root ganglion cells incubated with EAE splenocyte supernatant. Siponimod led to a dose-dependent reduction of EAE scores in chronic WT EAE. Using a suboptimal dosage of 0.45 µg/day, siponimod reduced clinical signs of EAE independent of BDNF-expression in immune cells in accordance with reduced infiltration and demyelination. Th and Tc cells in secondary lymphoid organs were dose-dependently reduced, paralleled with an increase of regulatory T cells. In vitro, neuronal viability trended towards a deterioration after incubation with EAE supernatant; siponimod showed a slight rescue effect following treatment of WT splenocytes. Neuronal gene expression for CCL2 and CX3CL1 was elevated after incubation with EAE supernatant, which was reversed after siponimod treatment for WT, but not for BNDFko. Apoptosis markers and alternative death pathways were not affected. Siponimod exerts both anti-inflammatory and neuroprotective effects, partially related to BDNF-expression. This might in part explain effectiveness during progression in MS and could be a target for therapy.

(Charles River, Wilmington, MA, USA) and partially BDNF-deficient mice on the same genetic background between 8 and 12 weeks were used for the experiments.Weight, age, and sex between groups were matched for each experiment.Partial BDNF-deficiency in mice (BDNF ko mice) was created with a Cre-loxP-model.Mice with a floxed BDNF fl/fl gene in the coding exon 8 22 were crossed with mice expressing Cre-recombinase controlled by lysM-Cre promoter specific for myeloid cells 23,24 and the CD4-Cre promoter specific for T-Helper cells 25 .Thus, BDNF ko mice have reduced BDNF expression in macrophages/microglia and T-Helper cells, both for infiltrating leukocytes as well as resident cells in lymphoid organs (Fig. S1) 23,26 .Phenotypically, BDNF ko mice show no clinical or behavioural impairment.
Mice were injected with 50-100 µg Myelin Oligodendrocyte Glycoprotein 35-55 (MOG 35-55 , Genosphere Biotechnologies, Paris, France) mixed with 200 µg complete Freund`s Adjuvant (CFA) subcutaneously into both hind flanks to induce experimental autoimmune encephalomyelitis (EAE) 21,23 .250 ng pertussis toxin (PTX, Sigma Aldrich GmbH, St. Louis, MO, USA) dissolved in PBS were injected intraperitoneally on days 0 and 2. Mice were treated with siponimod (MedChemExpress, South Brunswick Township, NJ, USA) in rape seed oil or vehicle daily via oral gavage in a blinded manner.Mice were weighted and scored daily using a 10-point-scale 21 .Mice with a score of 7 or a weight loss of > 20% were euthanized using cervical dislocation 27 .At the end of the experiment, mice were euthanised with carbondioxide inhalation according to AVMA guidelines 27,28 .Their blood, spleen, lymph nodes and spinal cord were extracted.Blood, spleen, and lymph node cells were prepared for flow cytometry.Histology and immunohistochemistry were performed with spinal cord slices and spleen.

Histochemistry for leukocyte infiltration and demyelination
Spinal cord was divided into cervical, thoracic, and lumbar parts.Spinal cord and spleen were fixated and embedded into paraffin.Slices were deparaffinized with Roti-Histol (Carl-Roth GmbH+ Co. KG, Karlsruhe, Germany) and dehydrated in a descending ethanol chain.
Pictures of slices were acquired with a Pecon® microscope (Carl Zeiss Microscopy GmbH, Oberkochen, Germany) using ZEN 3.3 (blue edition, version 3.3.89.0000,Carl Zeiss Microscopy GmbH, Oberkochen, Germany).For infiltration, the percentage of white matter area that was infiltrated by leukocytes, shown by their stained cell nuclei, was measured.For demyelination, the percentage of white matter stained by Schiff`s base instead of LFB was measured.To assess BDNF-knockout, the percentage of Iba1 + BDNF + or CD4 + BDNF + double positive cells in relation to Iba1 + or CD4 + single positive cells was manually counted and calculated.The percentages were then normalized to WT value.The images were blinded and analysed with ImageJ (version 1.535, Wayne Rasband and Co., national institute of health, USA).
Spleens from EAE mice 10 days post injection were explanted and sieved through 40 µm strainers.The splenocytes were separated via density gradient centrifugation with Ficoll® (Cytiva, Marlborough, MA, USA) and 2 × 10 5 lymphocytes were cultured on each 96-well plate in 200 µl splenocyte medium (Table S4).After 24 h, conditioned splenocyte supernatant was applied on DRG neurons in 96-well plates.After incubation of 24 h, viability and cell death analysis was performed for DRG neurons and splenocytes.Additionally, qPCR analysis and β-III-tubulin immunocytochemical staining was performed for DRG neurons.

Statistics
Data were analysed using Prism software V.9 (GraphPad Software, San Diego, CA, USA).Non-parametric data were analysed using Kruskal-Wallis test with post-hoc analysis using Dunn`s multiple comparisons test as shown in respective figure legends.Correlations were analysed using non-parametric Spearman correlation.Data are presented as mean values with standard error of the mean (SEM).A p-value < 0.05 was considered as statistically significant.

Siponimod improves clinical signs of experimental autoimmune encephalomyelitis
Across different studies, different applications and dosing regimens of siponimod in EAE have been conducted 3,30,31 .To assess the optimal dosage for treatment of BDNF ko mice, we treated C57BL6 wildtype (WT) mice prophylactically with three different dosages of 0.45 µg/day, 1 µg/day and 4 µg/day siponimod (Fig. 1a).As expected, siponimod lowered incidence by 66.7% in vehicle mice compared to 16.7% in the 4 µg group.This was reflected in a delay of clinical onset by 2 days in the two lower siponimod groups compared to the control group.In the 4 µg group, clinical signs only developed after day 47 (Fig. 1b).Siponimod also reduced overall disability as shown with clinical scores.At the end of the experiment, vehicle treated mice had a score of 2.5 ± 0.9 (mean ± SEM) compared to 0-0.66 in the treated groups (± 0-0.45) (p < 0.0001; Fig. 1c).Additionally, siponimod treated mice suffered less weight loss as a sign of general well-being (Fig. 1d).The dosage of 1 µg/day proved to be less effective at reducing EAE signs compared to the 0.45 µg group (p < 0.0001), which we attribute to one mouse from 1 mg/day group not benefitting from siponimod.Omitting the outlier showed similar beneficial effects of both dosages (Fig. S2).

Siponimod affects lymphocyte populations in blood, spleen and lymph nodes
Siponimod is well known to reduce lymphocyte circulation due to modulation of chemotaxis and hindering lymphocyte egress from lymph nodes 12 .Here, we investigated effects of siponimod on different immune sites including blood, spleen, and lymph nodes at the end of the experiment.We saw no effect on T cell frequencies (Fig. 2a).Regarding T-Helper cells (Th cells) and cytotoxic T cells (Tc cells), siponimod led to a decrease both in spleen (p = 0.0172 for Th cells, p = 0.0478 for Tc cells; Fig. 2b,c), and, for Th cells, in lymph nodes (p = 0.0093 for 4 µg/d vs. control, p = 0.0422 for 1 µg/day vs. control).Siponimod had no effect on frequencies of macrophages in blood, spleen and lymph nodes (Fig. 2d).B cell frequencies were increased in lymph nodes in the highest siponimod group compared to control while frequencies in blood and spleen were unaltered (p < 0.05; Fig. 2e).Subpopulations of Th and Tc cells are commonly differentiated into autoreactive IFN-y-positive (Th1 and Tc1 cells) and IL-17-positive T cells (Th17 and Tc17 cells) as well as protective regulatory T cells (Tregs).Siponimod did not induce a significant regulation regarding the frequencies of Th1 and Th17 populations (Fig. 2f,g).Tregs showed a dose-dependent reduction of frequencies in blood (p = 0.0017 for 4 µg/day vs. control; p = 0.0031 for 4 µg/day vs. 0.45 µg/day; Fig. 2h), and an increase in lymph nodes (p = 0.0087 for 4 µg/day vs. control).Surprisingly, siponimod did not induce any significant lymphocyte regulation in blood, except for Tregs.Tc1 and Tc17 cell populations were not altered (Fig. 2i,j), except for an increase of Tc1 cells in spleen (p = 0.0256 for 4 µg/day vs. 0.45 µg/day; Fig. 2i).

Siponimod improves clinical signs independent from BDNF in immune cells
For the investigation of siponimod in BDNF ko mice we used the suboptimal dosage of 0.45 µg/day, since this dosage elicited clear, but suboptimal clinical effects without suppressing EAE completely (Fig. 3a).Prophylactic treatment of BDNF ko mice following induction of EAE improved clinical signs significantly both for WT and BDNF ko mice compared to their respective controls (p < 0.0001 for WT and BDNF ko ; Fig. 3b).Interestingly, BDNF-deficiency had no effect on clinical scores (Fig. 3b).These results were corroborated by similar effects on weight loss.Control mice lost 9-13% of their starting weight at peak of disease, while siponimod treated mice lost significantly less weight (3-5%; p < 0.0001 for WT and BDNF ko ; Fig. 3c).Thus, BDNF-deficiency did not mitigate the positive clinical effect of siponimod in EAE.

Siponimod reduces immune cell infiltration and demyelination independent of BDNF in immune cells
To understand whether clinical effects were also corroborated histologically, we investigated immune cell infiltration and demyelination of cervical, thoracic and lumbar spinal cord.For vehicle groups, higher percentages of lumbar (Fig. 4b, 21.1-23.6%(± 7.4-4.3%))white matter were infiltrated compared to cervical parts (12.1-12.2%(± 3.3-4.1%);Fig. S3a).Analysis of lumbar spinal cord revealed a trend towards reduction of immune cell infiltration following siponimod treatment for WT mice and a significant decrease for BDNF ko mice (Fig. 4a,b).Likewise, demyelination trended to be reduced in siponimod treated mice in lumbar white matter.WT control mice and BDNF ko mice displayed 29.6 ± 4.6% and 36.3 ± 6.6% of demyelination in lumbar spinal cord, respectively.In contrast, siponimod treated mice showed 21.8 ± 6.9% demyelination for WT and 15 ± 5.8% for BDNF ko , reducing it by more than 50% in the latter case (Fig. 4c,d).For vehicle groups, the demyelinated white matter area increased towards caudal regions, being lowest in cervical area (21.7-26.5% (± 4-8.6%);Fig. S3b) and highest in lumbar area (Fig. 4d, 29.6-36.3%(± 4.5-6.6%)).There was a strong correlation of lumbar immune cell infiltration with disability as assessed using sum of scores (r = 0.77, p < 0.0001; Fig. 4e).Demyelination of white matter did not significantly correlate with sum of scores in mice (p = 0.08; Fig. 4f).However, immune cell infiltration strongly correlated with demyelination in lumbar spinal cord (r = 0.5068, p = 0.0226; Fig. 4g), as well as for cervical and thoracic parts (Fig. S3c).We also investigated alterations of immune cell subpopulations in blood, spleen, and lymph nodes via flow cytometry.Siponimod 0.45 µg/day had no effect on ratio of CD3-positive T cells, CD19-positive B cells and CD11b/F4/80-positive macrophages (Fig. S4).Th cells and their Th1, Th17 and Treg subpopulations showed no regulation in frequencies (Fig. S4).We saw decreased frequencies of Tc cells in spleen and lymph nodes for BDNF ko mice compared to WT mice (p = 0.0237 for spleen, p = 0.0125 for lymph nodes; Fig. S4c).Siponimod increased Tc1 ratio in lymph nodes (p = 0.0268; Fig. S4i) and reduced Tc17 frequencies in spleen (p = 0.0474; Fig. S4j).

Modulation of EAE splenocyte induced neurotoxicity by siponimod
To investigate effects of siponimod on the interaction between neurons and immune cells in absence of BDNF, we performed in vitro experiments using dorsal root ganglion cells (DRGs) and splenocytes from control, EAE vehicle and EAE siponimod treated mice.First, we assessed viability of splenocytes by measuring ATP quantity (Fig. 5a).Splenocyte viability was significantly more pronounced in WT EAE mice compared to WT control mice (p < 0.01; Fig. 5b).Siponimod treated splenocytes had similar viability compared to vehicle treated EAE mice (ns).Splenocytes from BDNF ko mice showed the same pattern as WT splenocytes.Thus, we suggest that both EAE induction and siponimod treatment activates splenocytes, increasing their viability.
We then incubated DRGs with splenocyte conditioned media for 24 h and then assessed neuronal viability (Fig. 5c).BetaIII tubulin staining showed the formation of neuronal networks for DRG cells (Fig. 5d).In this setup, we did not see significant changes, although there was a small trend suggesting that conditioned media from WT EAE splenocytes has harmful effects on DRGs (Fig. 5e).Siponimod administration seemed to have an beneficial influence on affected neurons, as viability seemed to improve for siponimod groups, independent of BDNF (all ns).

Siponimod shows BDNF-dependence regarding transcription of chemokines
We analysed the transcriptome of DRGs to understand the underlying mechanisms of neuronal loss following incubation with EAE conditioned media in vitro.We analysed apoptosis, which proceeds immunologically silently 32 .Neurons in contact with conditioned medium from EAE mice did not show genetic regulation of the most prominent apoptosis markers CASP3, the main effector caspase (Fig. 6a), and CASP9, which is an initiator caspase (Fig. 6b).Furthermore, we saw no effects on Caspase-3/-7 activity in these neurons (Fig. S5a).Additionally, we saw no differences in the transcription of cytochrome c, which is a mitochondrial protein of the respiratory chain that is ejected into the cytosol during apoptosis, the protein BAD, which is a representative of the BCL-2 family that modulates apoptosis, and Beclin 1, which serves as an autophagy signal and regulates apoptosis 33 (Fig. S6a-d).Therefore, transcription of typical apoptosis markers as well as Caspase-3/-7 activity are not affected by early interactions between neurons and lymphocytes.
We extended the analysis towards alternative death pathways and investigated pyroptosis, an inflammatory programmed cell death driven by Caspase 1. DRGs exposed to EAE medium had a slight, insignificant increase for the transcription of CASP1 in relation to control (Fig. 6c).Supernatant of siponimod treated WT splenocytes seemed to lower the neuronal CASP1 transcription only insignificantly.There was an inverse effect for BDNF ko , which rather showed an insignificant increase of CASP1 expression for siponimod groups.
Another alternative death pathway is necroptosis.Like pyroptosis, necroptosis is activated by Pathogen-associated molecular patterns (PAMPs) and the inflammatory protein TNF-α, which activates receptor-interacting  serine/threonine protein kinases 1 and 3 (RIPK1/RIKP3).RIPK3 transcription showed a trend towards elevation in neurons in the BDNF ko group despite siponimod administration (p = 0.1 for BNDF ko ctrl vs. BDNF ko EAE sip; Fig. 6d), but not for WT, indicating that BDNF-deficiency in key immune cells could have an effect on necroptosis.This is of special note, as BDNF shares the downstream target ERK1/2/MAPK with the necroptotic pathway 20,34 .This was in line with regulation of HMGB1, a marker for necrosis which further boosts inflammation (ns; Fig. 6e).TNF-α, which is a key inflammatory marker and initiator of necroptosis and pyroptosis, showed the same trends as RIPK3 (ns; Fig. 6f).
We analysed the genetic regulation of chemotactic markers CCL2 and CX3CL1.Neurons incubated with EAE splenocyte medium showed a significant activation of CX3CL1 transcription (p = 0.05 for WT, p = 0.0224 for BDNF ko ; Fig. 6g).Whilst CX3CL1 transcription was still significantly elevated for BDNF ko group despite siponimod treatment (p = 0.0426), we did not see the same significant increase for neurons in contact with siponimod-treated EAE splenocytes of WT mice (ns).This was similar for the regulation of CCL2, which showed an almost 4-fold transcription increase in neurons affected by EAE medium (p = 0.06 (ns) for WT, p = 0.0358 for BDNF ko ; Fig. 6h).Prior siponimod administration resulted in a non-significant, and less distinct, increase of CCL2 transcription.Thus, we deduce that siponimod might not be able to regulate the transcription of neuronal www.nature.com/scientificreports/In summary, siponimod showed no BDNF-dependency in vivo in EAE but possible BDNF-dependency regarding the modulation of neuronal inflammation in vitro.

Discussion
Multiple sclerosis progression remains a challenge for treating physicians since most medications effective in the treatment of RRMS yield ineffective results.The EXPAND study highlighted the S1PR-1 and -5 modulator siponimod as a putative tool in tackling SPMS 6 .Nevertheless, it remains unclear how siponimod might mediate neuroprotective effects when most other medications, including its predecessor fingolimod, failed.In this study, we investigated the importance of BDNF for the effects of siponimod in vivo and in vitro.Siponimod administered in chronic experimental autoimmune encephalomyelitis (EAE) ameliorated clinical signs of EAE.The suboptimal dosage of 0.45 µg/day had a marginal effect, which was not BDNF-dependent.We saw a reduction of immune cell infiltration into lumbar white matter area (Fig. 4b) for this siponimod dosage, and documented correlations between demyelination and immune cell infiltration into white matter (Fig. 4g).Higher siponimod dosages led to a retention of autoreactive immune cell populations in secondary lymphoid organs.In vitro, there were BDNF-dependent effects on transcription of certain inflammatory markers produced by neurons.
Siponimod reduces EAE scores due to its pleiotropic effects on lymphocyte egress, circulation and chemoattraction 3,5 .Furthermore, siponimod reduces microglial activation and induces axonal remyelination by inducing OPCs 13,14 .The effects of siponimod in EAE models have been displayed with various dosages and application methods 3,5,30 .Our aim was to mimic the daily oral intake of 1-2 mg/day by human beings, which approximates the dosage of 4-8 µg/day in mice 8,35 .Human equivalent dosages halted EAE disease activity completely, while lower dosages elicited alleviating effects, even for oral administration of the suboptimal dosage of 0.45 µg/day 3,30 .
In our study, we could define more closely how siponimod affects lymphocyte populations.As was reported previously by Gentile et al. 3 , who used intracerebroventricular administration, we saw no effects on peripheral lymphocyte count.Gergely et al. showed data which indeed shows reduction of absolute lymphocyte count, however, for human beings with dosages > 1 mg/day 5 .For human equivalent dosages, we saw an increase of B cell frequencies in lymph nodes, whereas Tc and Th cells were reduced in secondary lymphatic organs as shown previously using different mouse models 30 .Tregs exhibited a profound decrease in blood, and an increase in lymph nodes.Moreover, Treg frequencies negatively correlated with Th1 and Th17 frequencies in spleen.We postulate that the different effects of siponimod on Tregs as well as Th1 and Th17 in lymphoid organs may explain part of its beneficial immunomodulatory effects.This shift could be explained by the chemokine CCR7, which can be seen as the antagonist of S1P and holds back lymphocytes in lymph organs 36 .Thus, siponimod effects are more pronounced on CCR7-positive Th1 and Th17 cells than CCR7-negative Tregs.We assume that this shift of autoreactive to protective lymphocytes may play a role in the neuroprotective effect of siponimod.
Immunohistochemistry of spinal cord showed that siponimod reduces immune cell infiltration in lumbar white matter and suggests a reduction of demyelination, consistent with previous findings 11 .Of note, siponimod also reduces the size of meningeal ectopic lymphoid tissue 30 .Both these histological parameters are linked to disease severity but were not affected in the BDNF ko model used here.The generally accepted model proposes that lymphocyte infiltration causes demyelination, which, in turn, affects the motor functions of mice.This can explain why EAE symptoms predominantly begin in hind legs, as lumbar spinal cord shows more demyelination and leukocyte infiltration than cervical spinal cord.However, Frezel et al. argues lymphocyte infiltration to be a secondary effect, as neuronal production of the stress-associated transcription factor ATF3 precedes T cell infiltration 37 .Thus, we wanted to characterize the influence of infiltrating lymphocytes on neurons.
Effects of fingolimod are BDNF-dependent in hippocampal cultures, since treatment with BDNF-scavenging trkB receptor bodies diminishes fingolimod effects on neuronal dendrite growth and activation, questioning whether there might be an in vivo correlate 20 .We could show that siponimod reduces EAE scores and improves general well-being even in BDNF ko mice.Furthermore, we could show that EAE and siponimod administration affects splenocyte viability, although we could not clearly show the effect of these splenocytes on embryonal neurons.It is important to note that our Cre-loxP-model supported BDNF-deficiency on T cells and microglia/ macrophages.Although BDNF is also produced by neurons and glia such as astrocytes, activated immune cells are the major source of BDNF in active MS lesions 23,38 .Kerschensteiner et al. could show in vitro that this BDNFsecretion of immune cells provides protective effects on neuronal survival 39 , giving infiltrating immune cells a context-sensitive neuroprotective role.As we saw no aggravation of EAE symptoms in BDNF ko mice, autoreactive effects by infiltrating immune cells seem to be outdoing protective effects of immunological BDNF, relegating immunological BDNF to a secondary role; at least in the model used here.BDNF ko in further cell types could pertain a more severe EAE progress; however, more severe BDNF-deficiency also leads to aggressive behaviour, memory deficits and even early death in mice 40,41 .Therefore, our cell specific BDNF ko model provided an ethically acceptable level of genetic alteration, as mice did not show phenotypic abnormalities.There have been concerns about the specificity of lysM-Cre for macrophages/microglia 42 .Linker et al. were able to show that targeting lysM does not incur BDNF reduction in brain tissue 23 .
The lack of BDNF in key immune cells did not show an effect on the transcription of initiation markers for pyroptosis and necroptosis.These death pathways share the activation by PAMPs, DAMPs and TNF-α, and both serve, in contrast to typical apoptosis, as a potent amplifier of inflammation 34,43 .Both pathways have an executing protein complex.Pyroptosis is driven by inflammasomes containing CASP1, ASC and sensor markers such as NLRP1 or NLRP3.This leads to Gasdermin D-mediated pyroptotic bodies that cause neuroinflammation and CASP6-mediated demyelination 44,45 .MS medications such as cladribine and IFN-β have been shown to regulate pyroptosis 43 , which is unaffected by siponimod with the methods used here.In the case of necroptosis, necrosomes formed by RIPK1/RIPK3 activate the pro-necroptotic protein MLKL, which causes a caspase-8-independent cell death.Necroptosis has been described as a backup plan for cell death, as apoptosis and pyroptosis markers are capable of inhibiting necroptosis 32 .Additionally, RIPK1/3 leads to inflammation by a prolonged activation of ERK1/2/MAPK pathway 34 .This is noteworthy as ERK1/2 and akt phoshorylation are a target of BDNF as well and play a major role in counteracting cell death 20 .However, necroptosis leads to a prolonged activation of this pathway which turns this naturally protective and remyelinating pathway deleterious 46 .We hypothesize that ERK1/2 remains unnaturally activated by necroptosis as BDNF decreases.This might explain why BDNF-deficiency does not have a detrimental effect on EAE progress in our model, as the protective influence of the BDNF-ERK1/2 pathway might have been hijacked for inflammation by necroptosis, even in BDNF-rich environments.Yet, we cannot clearly show that siponimod prevents necroptosis and it remains an interesting topic for further research to investigate the link between BDNF and necroptosis.
Although a lack of BDNF in key immunomodulatory cells alone is not stressful for neurons, hinted by preserving their viability (Fig. 5e), it becomes stressful in neuroinflammatory environments that cannot be alleviated by siponimod, indicated by the increased transcription of the chemokines CX3CL1 and CCL2, produced by neurons upon inflammation which act on immune cells, especially microglia.
CX3CL1, also called fractalkine, is a neuronal transmembrane protein that can be cleaved to act as a soluble chemokine 47 .It attracts CX3CR1 expressing immune cells like microglia and T cells, maintaining inflammatory states 47 .However, the infiltrating CX3CR1-positive microglia elicit neuroprotection through phagocytosis and through ERK1/2 and akt signalling 48 , sharing the same pathway as BDNF.As BDNF and CX3CL1 share the same downstream target, it might be assumed that BDNF-deficiency is compensated by enhanced CX3CL1 expression.Above, we already discussed that a prolonged activation of the ERK1/2 pathway boosts inflammation.CCL2, also known as MCP-2, is a chemokine that mediates part of the chemoattractive effects of S1P.Upon binding S1PR-2, S1P enhances CCL2 secretion in neurons, which leads to an attraction and activation of CCR2 positive immune cells 49,50 .Additionally, CCR2 activated microglia are stimulated to express P2X7 receptor, which in turn leads to secretion of microglial BDNF 50 .This S1PR-2-CCL2-BDNF pathway has been argued to mediate neuroinflammation and demyelination.As we saw CCL2 modulation with a S1PR-1 and -5 modulator, CCL2 might also be modulated by those subunits.However, the lack of microglial BDNF in our BDNF ko mice and the CCL2 modulation did not reveal any particularly deleterious effects.We can support the hypothesis that the synthesis of both these neuronal chemokines increases during neuroinflammation but normalizes due to the S1PR-1 and -5 modulator siponimod, which also leads to less lymphocyte infiltration.As these chemokines remain elevated despite siponimod upon lack of BDNF in immune cells, we assume that protective effects of siponimod are mitigated in part by BDNF-deficiency.
In summary, we showed that treatment with siponimod ameliorates clinical signs of EAE.In vivo, clinical effects were not mediated by the presence of BDNF in immune cells.Siponimod, however, showed partial BDNF-dependency for modulation of key chemoattractive pathways.Further research may uncover the role and dependencies of BDNF in inflammatory pathways and alternative death pathways, presenting new targets for immune therapies.

Figure 1 .
Figure 1.Prophylactic siponimod dose-dependently ameliorates chronic MOG-EAE in C57BL6 mice.(a) 8-12 week old C57BL6 mice were immunized with MOG 35-55 and treated with PTX on days 0 and 2. Mice were treated with vehicle or different dosages of siponimod by oral gavage from day 0 to 50.Created with BioRender.com.(b) Incidence and (c) clinical scores showed a significant reduction for siponimod treatment in all groups compared to vehicle.(d) Weight loss was significantly lower in 0.45 µg/day and 4 µg/day siponimod treated mice compared to the vehicle group.n = 6 for each group.Data are shown as mean ± SEM.Weight was normalized to respective group mean.Incidence data (b) were analyzed with Logrank test.Score and weight data (c, d) were analyzed for normality with Shapiro-Wilk test and for significance with Kruskal-Wallis test with posthoc analysis using Dunn's multiple comparisons test.Significances are depicted as *p < 0.05; ***p < 0.001; ****p < 0.0001.WT wildtype, MOG Myelin oligodendrocyte glycoprotein, s.c.subcutaneously, PTX pertussis toxin, i.p. intraperitoneally.

Figure 2 .
Figure 2. Siponimod dose-dependently modifies the T cell compartment towards a regulatory state.Cells from blood, spleen and lymph nodes of siponimod or vehicle-treated EAE mice were analyzed via flow cytometry.T cell subpopulations were related to parent.(a) Whereas T cells showed no regulation due to siponimod treatment, there was (b) a reduction of frequencies in spleen and lymph nodes for Th cells and (c) in spleen for Tc cells.(d) Macrophage populations were not affected.(e) B cells in blood and spleen were unaffected, while showing an increase in lymph nodes.(f, g) Th1 and Th17 populations did not show significant changes.(h) Siponmod led to a dose-dependent increase in Treg cell frequenecy in blood, and a decrease in lymph nodes.(i) Tc1 cell frequency was elevated in spleen, and (j) Tc17 frequency showed no change for siponimod treatment.(k, l) Th1 and Th17 cells were restrained dose-dependently in spleen, showing an inverse correlation with Treg frequencies.n = 6 for each group.Data are shown as mean ± SEM.Flow cytometry data (a-j) were analyzed for normality with Shapiro-Wilk test and for significance with Kruskal-Wallis test with post-hoc analysis using Dunn`s multiple comparisons test.Correlation analysis (k, l) was performed using Spearman correlation and shown with 95% confidence bands.Significances are depicted as *p < 0.05; **p < 0.01.Th cells T helper cells, Tc cells cytotoxic T cells, Treg cells/Tregs T regulatory cells.

Figure 3 .
Figure 3. Treatment with siponimod ameliorates MOG-EAE in control and BDNF ko mice.(a) MOGimmunized WT and BDNF ko mice (8-12 weeks) were administered with 0.45 µg/d siponimod or vehicle daily for 50 days.Created with BioRender.com.(b) Clinical scores were ameliorated and (c) weight loss was averted in siponimod treated mice independent of BDNF ko .Shown are pooled data of n = 3 independent experiments.WT vehicle: n = 15, WT siponimod: n = 17, BDNF ko vehicle: n = 15, BDNF ko siponimod: n = 18.Scores of four mice without EAE-symptoms (WT vehicle: n = 2, WT siponimod: n = 1, BDNF ko vehicle: n = 1) and three mice with premature exitus (BDNF ko vehicle: n = 3).were excluded.Data are shown as mean ± SEM.Weight was normalized to group mean.Data were analyzed for normality with Shapiro-Wilk test and tested for significance with Kruskal-Wallis test with post-hoc analysis using Dunn's multiple comparisons test.Significances are depicted as ****p < 0.0001.BDNF ko = partial BDNF knock-out.

Figure 4 .
Figure 4. Siponimod treatment reduces lymphocyte infiltration of white matter in lumbar spinal cord.(a) Represenative hematoxylin and eosin (HE) images of spinal cord sections.(b) BDNF ko EAE mice showed less lumbar lymphocyte infiltration when administered with 0.45 µg siponimod daily, which is also suggested for WT mice.(c) Represenative LFB images of spinal cord sections.(d) There is no significant reduction of demyelination for lumbar white matter caused by 0.45 µg/day siponimod independent of BDNF.(e) High disease activity strongly correlated with higher lymphocyte infiltration in respective mice.(f) Higher degress of demyelination for lumbar white matter area seemed to be linked with sum of scores, yet missing significance narrowly.(g) Mice with more infiltration showed a trend towards more demyelination in lumbar spinal cord, missing significance.Lumbar sections of spinal cord were analyzed in quadruplicates for each mouse.WT vehicle: n = 4, WT siponimod: n = 6, BDNF ko vehicle: n = 4, BDNF ko siponimod: n = 6.Two mice without EAE symptoms (WT vehicle: n = 2) and two mice with premature exitus (BDNF ko vehicle: n = 2) were excluded.Data are shown as mean ± SEM.Infiltration and demyelination data (b, d) were analyzed for normality with Shapiro-Wilk test and tested for significance with Kruskal-Wallis test with post-hoc analysis using Dunn`s multiple comparisons test.Correlation analysis (e-g) was performed using Spearman correlation and 95% confidence bands are depicted.Significances are depicted as *p < 0.05.Scale bars: 500 µm.HE hematoxylin & eosin staining, LFB Luxol's Fast Blue staining.

Figure 5 .
Figure 5. Analysis of neurotoxicity induced by EAE conditioned media of splenocytes.(a) Splenocytes from mice 10 d post immunization were cultured and conditioned media were harvested 24 h after medium change.Created with BioRender.com.(b) MOG-immunisation and siponimod treatment led to a BDNF-independent increase of splenocyte viability, using an ATP-Glo assay.(c) Embryonal DRGs were incubated for 24 h with conditioned media of splenocytes.(d) Represenative βIII-tubulin staining of DRG networks.(e) Neurons incubated with conditioned media of EAE splenocytes show a trend towards reduction of viability, measured by using an ATP-Glo assay, which can be halted by prior siponimod administration of EAE mice independent from BDNF.For viability (b, e), n = 5 experiments with n = 2 mice in each group are presented.WT control: n = 10, WT EAE vehicle: n = 10, WT EAE siponimod: n = 8, BDNF ko control: n = 9, BDNF ko EAE vehicle: n = 10, BDNF ko EAE siponimod: n = 8.For splenocytes (b), 3 to 4 replicates and for neurons (e), 8-12 replicates are presented.Data were normalized to their respective WT control group and shown as mean ± SEM.Data (b, e) were tested for normality with Shapiro-Wilk test and tested for significance with Kruskal-Wallis test with post-hoc analysis using Dunn's multiple comparisons test.Significances are depicted as **p < 0.01, ***p < 0.001.Scale bar: 100 µm.EAE experimental autoimmune encephalomyelitis, DRG dorsal root ganglion cell.